Screening assays for agonists and antagonists of receptor activator of NF-κB

Abstract

The present invention provides methods for screening for a molecule that antagonizes or agonizes RANK activity. One aspect of the invention involves the growth of RANK responsive cells in semi-solid medium, wherein exposure to a RANK antagonist promotes colony formation. Other aspects of the invention rely on promoter/reporter constructs using RANK responsive promoters derived from the MMP-9 and TRAP genes. Additional aspects of the invention exploit the ability of RANK to activate c-src activity, F-actin ring formation and CaPO 4 resorption.

1. A method for screening for a molecule that agonizes or antagonizes RANK activity in cells expressing RANK, wherein RANK is a polypeptide that is capable of activating NF-κB and that is selected from the group consisting of: i) amino acids 1-616 of SEQ ID NO:2; ii) amino acids 1-625 of SEQ ID NO:4; and iii) a polypeptide having an at least 90% amino acid sequence identity with (i) or (ii), said method comprising the steps of:
 (a) contacting a candidate molecule with cells expressing RANK, said RANK expressing cells comprising a nucleic acid molecule encoding a reporter protein, said nucleic acid molecule being operably linked to a RANK-responsive regulatory nucleic acid sequence that is selected from the group consisting of a human or murine MMP-9 promoter and a human or murine TRAP promoter, wherein the cells are exposed to a RANK trigger prior to, during or after being contacted with said candidate molecule;  
 (b) determining whether the level of reporter molecule expression in the contacted cells is enhanced or reduced as compared with the level of reporter molecule expression in a culture of reference RANK expressing cells that are not contacted with the candidate molecule; and  
 (c) identifying the candidate molecule as a RANK agonist if the level of reporter molecule expression in the contacted cells is comparatively enhanced and identifying the candidate molecule as an RANK antagonist if the level of reporter molecule expression in the contacted cells is comparatively reduced.  
 
     
     
       2. The method of  claim 1 , wherein RANK is triggered in the RANK expressing cells by a method selected from the group consisting of:
 (a) contacting the cells with a RANK-L polypeptide, wherein the RANK-L polypeptide comprises amino acids 162-317 of SEQ ID NO:6;  
 (b) contacting the cells with an agonistic anti-RANK antibody;  
 (c) contacting the cells with cells that express RANK-L, wherein RANK-L is a polypeptide that comprises amino acids 162-317 of SEQ ID NO:6; and  
 (d) overexpressing RANK in said RANK expressing cells.  
 
     
     
       3. The method of  claim 2 , wherein RANK is triggered in the RANK expressing cells by contacting the cells with a RANK-L polypeptide comprising amino acids 162-317 of SEQ ID NO:6 , and further wherein said RANK-L polypeptide is selected from the group consisting of native RANK-L, a leucine zipper fusion of RANK-L, and a FLAG polyHis fusion of RANK-L. 
     
     
       4. The method of  claim 1 , wherein the RANK responsive regulatory nucleic acid is a MMP-9 promoter. 
     
     
       5. The method of  claim 4 , wherein the MMP-9 promoter comprises nucleotides 1769-3591 of SEQ ID NO:11. 
     
     
       6. The method of  claim 1  wherein the step of contacting RANK expressing cells with a candidate molecule comprises expressing in said cells an introduced DNA molecule that encodes a candidate nucleic acid molecule or a candidate protein molecule. 
     
     
       7. The method of  claim 6  wherein said introduced DNA molecule is a cDNA molecule. 
     
     
       8. The method of  claim 6  wherein said introduced DNA molecule is integrated into the genome of said cells. 
     
     
       9. The method of  claim 6  wherein said introduced DNA molecule is not integrated into the genome of said cells. 
     
     
       10. The method of  claim 6  wherein said candidate molecule is a protein encoded by the introduced DNA molecule. 
     
     
       11. The method of  claim 6  wherein said candidate molecule is a nucleic acid molecule encoded by the introduced DNA molecule. 
     
     
       12. The method of  claim 11  wherein said nucleic acid molecule possesses ribozyme activity. 
     
     
       13. The method of  claim 6 , further comprising isolating said introduced DNA molecule from a colony formed from said contacted cells. 
     
     
       14. The method of  claim 1 , wherein the step of contacting RANK expressing cells with a candidate molecule comprises culturing said cells in the presence of the candidate molecule, and further wherein said candidate molecule is a protein. 
     
     
       15. The method of  claim 1 , wherein the step of contacting RANK expressing cells with a candidate molecule comprises culturing said cells in the presence of said candidate molecule. 
     
     
       16. The method of  claim 1 , wherein said reporter molecule is selected from the group consisting of luciferase, green fluorescent protein, alkaline phosphatase and a heterologous surface protein. 
     
     
       17. The method of  claim 16 , wherein the reporter molecule is a heterologous surface protein that is selected from the group consisting of human IL-2 receptor, murine IL-4 receptor, human CD2 protein, human CD4 protein, human CD8 protein, luciferase protein, β-galactosidase, and green fluorescent protein. 
     
     
       18. The method of  claim 1 , wherein said RANK expressing cells express a defective RANK molecule and wherein said screening is for an agonist that complements said defective RANK activity. 
     
     
       19. The method of  claim 1 , wherein said RANK expressing cells are hematopoietic cells. 
     
     
       20. The method of  claim 1 , wherein said RANK expressing cells are RAW 264.7 cells. 
     
     
       21. The method of  claim 1 , further comprising the step of purifying the candidate molecule from cells in which the level of expressed reporter molecule is determined to be comparatively enhanced or reduced. 
     
     
       22. The method of  claim 1 , wherein the level of reporter molecule expression is determined by an assay selected from the group consisting of a fluorescence-based assay, a solid phase assay, and an assay employing a radioactive compound. 
     
     
       23. The method of  claim 1 , wherein determining the level of expressed reporter molecule comprises physically isolating by fluorescence-based cell sorting the cells that are expressing said reporter molecule. 
     
     
       24. A method according to  claim 1 , wherein the MMP-9 promoter is a murine promoter comprising nucleotides 1769-3591 of SEQ ID NO:11 and the TRAP promoter is a murine promoter comprising nucleotides 1-1991 of SEQ ID NO:12. 
     
     
       25. A method for screening for a molecule that agonizes or antagonizes RANK activity, said method comprising the steps of:
 (a) contacting a candidate molecule with cells expressing a defective RANK polypeptide consisting of amino acids 1-622 of SEQ ID NO:10, said cells comprising a nucleic acid molecule encoding a reporter protein, said nucleic acid molecule being operably linked to a RANK-responsive regulatory nucleic acid sequence that is selected from the group consisting of a human or murine MMP-9 promoter and a human or murine TRAP promoter;  
 (b) determining whether the level of reporter molecule expression in the contacted cells is enhanced or reduced as compared with the level of reporter molecule expression in a culture of reference cells expressing said defective RANK and comprising said nucleic acid molecule that are not contacted with the candidate molecule; and  
 (c) identifying the candidate molecule as a RANK agonist if the level of reporter molecule expression in the contacted cells is comparatively enhanced and identifying the candidate molecule as a RANK antagonist if the level of reporter molecule expression in the contacted cells is comparatively reduced.